RESUMEN
The cellular prion protein (PrP(C)) is a conserved glycoprotein predominantly expressed in neuronal cells. Its purpose in living cells is still enigmatic. To elucidate on its cellular function, we performed a yeast two-hybrid screen for interactors. We used murine PrP(C) (amino acids 23-231) as bait to search a mouse brain cDNA expression library. Several interaction partners were identified. Three of them with a high homology to known sequences were further characterized. These candidates were the neuronal phosphoprotein synapsin Ib, the adaptor protein Grb2, and the still uncharacterized prion interactor Pint1. The in vivo interaction of the three proteins with PrP(C) was confirmed by co-immunoprecipitation assays with recombinant and authentic proteins in mammalian cells. The binding regions were mapped using truncated PrP constructs. As both synapsin Ib and Grb2 are implicated in neuronal signaling processes, our findings further strengthen the putative role of the prion protein in signal transduction.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Cricetinae , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/metabolismo , Exorribonucleasas , Proteína Adaptadora GRB2 , Biblioteca de Genes , Aparato de Golgi/metabolismo , Immunoblotting , Ratones , Microsomas/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos/metabolismo , Mutación Puntual , Proteínas PrPC/fisiología , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/metabolismo , ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Sinapsinas/metabolismo , Distribución Tisular , Transfección , Células Tumorales Cultivadas , Técnicas del Sistema de Dos Híbridos , Virus Vaccinia/genéticaRESUMEN
Prion diseases are fatal and transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrP(c)). We show that the chemical compound Suramin induced aggregation of PrP in a post-ER/Golgi compartment and prevented further trafficking of PrP(c) to the outer leaflet of the plasma membrane. Instead, misfolded PrP was efficiently re-routed to acidic compartments for intracellular degradation. In contrast to PrP(Sc) in prion-infected cells, PrP aggregates formed in the presence of Suramin did not accumulate, were entirely sensitive to proteolytic digestion, had distinct biophysical properties, and were not infectious. The prophylactic potential of Suramin-induced intracellular re-routing was tested in mice. After intraperitoneal infection with scrapie prions, peripheral application of Suramin around the time of inoculation significantly delayed onset of prion disease. Our data reveal a novel quality control mechanism for misfolded PrP isoforms and introduce a new molecular mechanism for anti-prion compounds.
Asunto(s)
Proteínas PrPSc/biosíntesis , Enfermedades por Prión/prevención & control , Priones/efectos de los fármacos , Sarcosina/análogos & derivados , Suramina/uso terapéutico , Ácidos , Amidohidrolasas/metabolismo , Animales , Compartimento Celular , Detergentes/farmacología , Aparato de Golgi/metabolismo , Líquido Intracelular/metabolismo , Ratones , Ratones Transgénicos , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Priones/genética , Priones/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína , Sarcosina/farmacología , Suramina/farmacología , Células Tumorales Cultivadas , Red trans-Golgi/metabolismoRESUMEN
We describe the shortest prion protein allele known to date. Surprisingly, it is found as a polymorphism exactly in a species (prosimian lemurs) which seems highly susceptible to oral infection with BSE-derived prions. The truncation of the prion protein we found raises several questions. First, is the truncated octarepeat structure we describe, consisting of two octarepeats, still functional in copper binding? A second question is whether this truncation is related to the remarkable oral infectibility of lemurs with BSE-derived prions. And finally, one could argue that this genotype alone might favour development of a prion disease, even in the absence of exogenous infection.